Engineered nucleases, including zinc finger nucleases, TALENs, CRISPR/Cas nuclease systems, Ttago nucleases and homing endonucleases designed to specifically bind to target DNA sites are useful in genome engineering. For example, zinc finger nucleases (ZFNs) and TALENs (including TALENs comprising Fok1-TALE DNA binding domain fusions, Mega TALs and cTALENs) are proteins comprising engineered site-specific zinc fingers or TAL-effector domains fused to a nuclease domain. Such nucleases have been successfully used for genome modification in a variety of different species at a variety of genomic locations. See, for example, See, e.g., U.S. Pat. Nos. 8,623,618; 8,034,598; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983, 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes.
Cleavage of a target nucleotide sequence by these nucleases increases the frequency of homologous recombination (HR) with a donor at the targeted locus by more than 1000-fold. Homology-directed repair (HDR) of a nuclease-mediated cleavage event can be used to facilitate targeted insertion of a gene (transgene) by co-delivering a donor molecule encoding a gene flanked by sequence homologous to region surrounding the break site. In addition, the repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene modification, including gene (transgene) insertion by NHEJ-dependent end capture. See, e.g., U.S. Patent Publication No. 20110207221. In addition to targeted integration of a transgene, nuclease-mediated cleavage and repair by NHEJ can result in non-specific insertions and/or deletions (“indels”) at the site of the break. Thus, nucleases specific for the targeted region can be utilized such that the transgene construct is inserted by either HDR- or NHEJ-driven processes, or for knockout of a gene through error-prone NHEJ repair of the nuclease-mediated DSB. Gene correction may also be accomplished using targeted nucleases and donor molecules designed to replace a specified region in an endogenous gene with sequences supplied in the donor. A specific double strand break (DSB) is introduced in the gene and in the presence of the gene correcting donor DNA, the sequences of interest are replaced using those of the donor via homology dependent recombination.
This nuclease-mediated targeted transgene insertion approach offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches, since it allows exact transgene positioning to minimize the risk of gene silencing or activation of nearby oncogenes. However, efficiency of nuclease activity can be influenced by a variety of factors such as accessibility of the chromosomal DNA target and the quality of the binding interaction between the nuclease and its target nucleic acid. Efficiency of these approaches in vivo is further complicated by factors such as target tissue accessibility and tissue uptake of vectors that deliver the nucleases and transgene donors, and nuclease expression levels that can be achieved in vivo. To increase the success rate of nuclease driven genomic modifications, researchers often have to resort to introducing selectable markers during donor integration in order to be able to select variants that have had modifications from those that have not been modified (see, for example, U.S. Pat. No. 6,528,313). For a number of applications, use of selectable markers is not desirable as this technique leaves an additional gene or nucleic acid sequence inserted into the genome.
Thus, there remains a need for compositions and methods for increasing nuclease-mediated targeted integration of transgenes to allow for even more efficient use of these powerful tools.